The goal of the investigations described in this proposal is to obtain detailed information on the natural history of the T cell response to islets as it develops in islet infiltrates of pre-diabetic NOD mice In this analysis we will exploit our recent finding that insulin-specific T cells are a predominant component of islet-specific T cells and focus on this insulin-specific response. The experiments are designed to test four hypotheses. i. The frequency of insulin-specific T cells within populations of T cells presents in islet infiltrates is low at the early stages of beta cell damage and these T cells are of limited TCR diversity. ii. As the degree of infiltration and beta cell damage increases the frequency of insulin-specific T cells, as a sub population of islet infiltrates, increases and the TCR diversity of the insulin-specific T cells present in islet infiltrates increases. iii. Individual pre-diabetic NOD mice at similar stages of disease progression will employ the same TCR repertoire in the response to insulin and islet antigens. iv. Islet-specific and insulin-specific T cells isolated from islet infiltrates will be of the Th 1 phenotype. Testing the above four hypotheses will require isolation and characterization of panels of islet-specific T cells clones from individuals pre-diabetic mice at successive intervals in disease progression. The preliminary results presented in this proposal demonstrate the proficiency of our laboratory in the routine isolation of islet-specific T cells from islet infiltrates of young, pre-diabetic mice. There are four specific aims. i. Isolation of islet-specific an insulin-specific T cells from islet infiltrates of pre-diabetic NOD mice at various stages of disease progression, in-vitro characterization of these cells with regard to CD4/CD8 phenotype and establishment of islet-specific and insulin- specific T cell lines and clones from these cells. ii. Determine the kinetics of appearance of insulin-specific T cells in islet infiltrates by a comprehensive limiting dilution frequency analysis of individual NOD mice in age matched groups at one week age intervals. iii. In vitro characterization of the T cells isolated from islet infiltrates for antigen-specificity, lymphokine production profiles, and sequence analysis of the TCR a/B chains by PCR techniques. iv. Assessment of the capacity of insulin-specific T cell lines and clones to: a, induce islet damage upon adoptive transfer into NOD- scid/scid mice; b, synergies with known diabetogenic T cell clones in adoptive transfer of diabetes; c, inhibit adoptive transfer of diabetes by known diabetogenic T cell clones.